The present invention pertains to a set of devices and their related methods to rapidly and simply perform detection, quantitation, and identification of microorganisms in samples collected from a variety of sources including but not limited to drinking water, wastewater, industrial and pharmaceutical process waters or surface extraction solutions.
Diverse Needs & Requirements
There are diverse needs for microbiological tests and a diverse array of test methods and devices to assess their presence. In the US, municipal drinking water suppliers are required by the Environmental Protection Agency (EPA) to test for the presence of one E. coli or Total Coliforms in 100 mL of their product water. In Europe, tests require both identification and enumeration of these organisms' presence. Recreational water monitoring requires enumeration of E. coli and fecal coliforms from fresh water and Enterococcus faecalis from salt water with the United States Congress mandating detection in less than 4 hours. Protection of the food supply chain involves evaluating critical points in the preparation process where contamination is most likely to occur and where it will have the greatest impact. In these applications, no one test format is generally useful, and a need for greater test speed would benefit the ultimate application utility.
To meet the diverse needs of these applications, a variety of methods have been developed. Most notably, there is direct filtration and growth of the bacteria on a membrane, growth of bacteria in a solution and observation of a chemical change arising from their growth, and direct observation of the bacteria or some property of the bacteria in a solution using a developing agent. An ideal test method would be:                simple to perform with a minimal number of handling steps        capable of analyzing a sample of a milliliter or less or conversely up to a liter in volume        sensitive to as few as one organism        able to analyze more than one specific type of organism within a given sample        able to detect and measure specific types of target organisms in the presence of large numbers of non-target organisms        able to differentiate that organism(s) are alive and replicating rather than senescent or dead        able to quantify the viable organisms present        highly accurate with a low rate of false positives/false negative results        able to complete an analysis and provide results within hours        executable in an outdoor field environment, not requiring a laboratory or ancillary support equipment        able to provide test results without requiring operator involvement after the test is initiated        executable by minimally skilled operators        small, portable, low power, and self-contained        
Direct observation of cells can be accomplished using a variety of methods as simple as turbidity or more sophisticated and specific methods using labeled antibodies directed at specific antigens on the cell surface. Using agglutination methods or lateral flow technology, the antigen-antibody method can be rapid, performed in the field and easily read by non-technical personnel. However, large numbers of organisms are typically required, and the methods are unable to differentiate viable from non-viable organisms. DNA technology can similarly provide a relatively rapid result. However, these techniques require highly trained personnel manipulating samples in a laboratory to achieve reproducible results, and it is still not possible to differentiate viable from non-viable organisms.
Filtration allows for concentration of a large volume water sample which can then be incubated with a number of different media to allow growth and identification of viable organisms. Detailed materials and methods are published in Standard Methods for the Examination of Waste and Wastewater. In these methods, a sample is typically filtered using a filter membrane held in a filter funnel. After filtration, the membrane is removed from the funnel, transferred to a culture plate containing growth media where it is incubated for typically for 24-48 hours. After the incubation period, colonies growing on the membrane can be identified through morphological-, biochemical-, antigenic- or DNA-fingerprinting analyses. The principle advantages of membrane filtration methodology are its ability to concentrate organisms from large volume samples and its ability to identify and quantify specific organisms from the sample. Obvious disadvantages of this method are the need to handle the membrane, which increases the potential for contamination, and the long incubation time. Devices have been contrived that minimize the need for filter handling after sample processing by integration of the filter membrane with a filter funnel and culture plate (e.g., Millipore, 55-Monitor); however, typical analysis times still require 24 hours of incubation.
In some applications (e.g., US drinking water tests), only a determination of the presence or absence of a viable indicator organism is required. Edberg [U.S. Pat. No. 5,429,933] discloses a simple assay for both the detection of coliforms and E. coli through the use of labeled metabolites and a highly enriched medium to promote the growth of injured microorganisms. The assay requires the addition of a prescribed volume of water to a vessel containing the dried culture medium and labeled metabolites. The vessel is incubated at the appropriate temperature for 18 hours, and the vessel is read for the presence of fluorescence and color to determine the presence of coliforms and/or E. coli. 
The obvious advantage of this method is its simplicity; however, the method does not provide for quantification of the contamination level and still requires 18 hours of incubation. Other disadvantages to the method include: a narrow time window (18-26 hours) during which the sample must be observed; indeterminate color changes or obfuscation of the color by the sample's own color (e.g., iron containing water); the need to provide for a large incubator space and a waterbath (or other device with high heat transfer capability) for preheating the 100 mL of sample water to the required temperature in a short period of time.
Several workers in the field have proposed the use of an optical instrument to read changes in the optical indicator. Fiksdal (“Monitoring of fecal pollution in costal waters by use of rapid enzymatic techniques.” Appl. Environ. Microbiol. 60:1581-84) proposed use of a fluorometer in 1994 to measure color changes in a sample resulting from β-galactosidase. In a related patent, Øfjord (U.S. Pat. No. 5,972,641) similarly proposed the use of a fluorometer to measure a sample's fluorescence after incubation for a prescribed time. In this method, a small aliquot of sample fluid was mixed with a growth media containing 4MU-galactopyranoside which is hydrolyzed by coliform bacteria to produce a fluorescent indicator of coliform activity. After 9 hours of incubation, an aliquot of the sample-media mixture is transferred to a cuvette, alkalinized with 2.5M NaOH then measured in a carefully calibrated fluorometer. If the sample's fluorescence is greater than a prescribed level, the sample is considered to be positive.
Øfjord's approach was later semi-automated in the Colifast CA-100 (Colifast Systems ASA). Methods and results using this system are reported by Angles (“Field evaluation of a semiautomated method for rapid and simple analysis of recreational water microbiological quality.” Appl. Env. Microbiol. 66(10):4401-07). The instrument requires dedicated floor space, requires careful calibration against known fluorescent standards and requires measurement of a blank sample to estimate a detection threshold. Samples of 6 mL water (maximum sample size) were mixed with 6 mL concentrated media then incubated for 7 hours. Using a series of tubing and pumps, the machine withdraws a small aliquot every 100 minutes, adds NaOH to alkalinize the sample then measures its fluorescence. If the sample fluorescence exceeds the threshold level, it is considered positive. The test showed that highly contaminated samples could be rapidly assessed. However, the system was prone to cross-contamination from previous samples and it had a high rate of false positives, presumably due to background enzyme activity. Further, it was not possible to test larger sample volumes (e.g. >100 mL) which is needed to meet drinking and bottled water testing requirements.
Famleitner (“Rapid enzymatic detection of Escherichia coli contamination in polluted river water.” Lett Appl. Micro. 33:246-50) describes using a filter to capture and concentrate a sample followed by measurement of fluorescence. In this work, the processed filter is removed from the filter funnel and added to a beaker containing growth media with the fluorescent-conjugate 4-methylumbelliferyl-β-glucuronide (4MU-glu) and incubated at 37° C. Aliquots were removed, alkalinized and measured for increases in fluorescence after 10 minutes, 20 minutes, and 30 minutes on a sophisticated benchtop fluorometer. Comparison with sample blanks and known fluorescence standards allowed the authors to quantify the rate of 4-MU production and subsequently to demonstrate a log-log relationship between the glucuronidase activity and the number of viable E. coli for organism concentrations greater than 1000 cfu/100 mL. No attempt to quantify below this level was reported. Consequently, although the method was fast, it had a number of drawbacks including its a lack of sensitivity, laborious handling requirements, requirements for a lab based fluorometer with both positive and negative standards and a high likelihood for false positives at low contamination levels of E. coli. 
Thus, a variety of prior art methods and devices have been devised to meet some testing requirements for the environmental, food, pharmaceutical and research communities. In general, they are laborious, prone to contamination, or they provide very limited information about the sample. Tests that measure viability of organisms typically require 24 hours of incubation before they can be interpreted. Faster tests will typically not distinguish between viable and non-viable organisms. Additionally, the faster tests require a large and expensive lab-based instrument which requires extensive calibration and testing of negative controls.
Accordingly, current practices and described art have shortcomings with respect to meeting the attributes of an ideal test as identified previously. There is a need in the art for a method and device that integrates the benefits of (i) membrane filtration in order to analyze large volume samples, (ii) incubation and culture of organisms in order to differentiate live cells and to expand the target from potentially a single cell to the level of a reliably measurable population, (iii) a single small disposable device that is easily prepared for analysis with minimal risk of contamination, and (iv) an automated detection method to rapidly identify indications of sample contamination by more than one type organism and quantify the numbers of organisms.